Fungicides have myriad uses, including for crop protection; as food, feed, and cosmetics preservatives; and as therapeutic agents for both human and veterinary applications. Crop yield reduction, foodborne diseases and fungal infections of both humans and animals are a problem in both developed and developing countries.
Synthetic insecticides or fungicides often are non-specific and therefore can act on organisms other than the target organisms, including other naturally occurring beneficial organisms. Because of their chemical nature, they may also be toxic and non-biodegradable. Consumers worldwide are increasingly conscious of the potential environmental and health problems associated with the residuals of chemicals, particularly in food products. This has resulted in growing consumer pressure to reduce the use or at least the quantity of chemical (i.e., synthetic) pesticides. Thus, there is a need to manage food chain requirements while still allowing effective pest control.
A further problem arising with the use of synthetic insecticides or fungicides is that the repeated and exclusive application of an insecticide or fungicide often leads to selection of resistant pathogenic microorganisms. Normally, such strains are also cross-resistant against other active ingredients having the same mode of action. This eliminates effective control of the pathogens with the active compounds. However, active ingredients having new mechanisms of action are difficult and expensive to develop.
The risk of resistance development in pathogen populations as well as environmental and human health concerns have fostered interest in identifying alternatives to synthetic insecticides and fungicides for managing plant diseases. The use of biological control agents is one alternative.
Non-ribosomal peptides, such as the fusaricidins, are well-recognized for their antimicrobial properties and have been used in the field of crop protection. Because of their mode of action, they also have potential uses in biopharmaceutical and other biotechnology applications. Fusaricidins can be isolated from Paenibacillus sp. and have a ring structure composed of 6 amino acid residues in addition to 15-guanidino-3-hydroxypentadecanoic acid. Fusaricidins isolated from Paenibacillus polymyxa include LI-F03, LI-F04, LI-F05, LI-F07 and LI-F08 (Kurusu K, Ohba K, Arai T and Fukushima K., J. Antibiotics, 40:1506-1514, 1987) and additional fusaricidins A, B, C and D have been reported (Kajimura Y and Kaneda M., J. Antibiotics, 49:129-135, 1996; Kajimura Y and Kaneda M., J. Antibiotics, 50:220-228, 1997).
Certain fusaricidins are known to have germicidal activity against plant pathogenic fungi such as Fusarium oxysporum, Aspergillus niger, Aspergillus oryzae and Penicillium thomii. Some fusaricidins also have germicidal activity against Gram-positive bacteria including Staphylococcus aureus (Kajimura Y and Kaneda M., J. Antibiotics, 49:129-135, 1996; Kajimura Y and Kaneda M., J. Antibiotics, 50:220-228, 1997). In addition, it has been found that specific fusaricidins have antifungal activity against Leptosphaeria maculans which causes black root rot of canola (Beatty P H and Jensen S E., Can. J. Microbiol., 48:159-169, 2002).
There is a need to identify efficient methods of enriching active compounds such as fusaricidins in fermentation broths. This can be particularly difficult as such fermentation broths often contain high levels of exopolysaccharides (EPS) and other biopolymers that increase the viscosity of the broth and thereby make the enrichment process more challenging.
EPS are high-molecular-weight polymers that are composed of sugar residues and are secreted by many microorganisms into the surrounding environment. Microorganisms synthesize a wide spectrum of multifunctional polysaccharides including intracellular polysaccharides, capsular polysaccharides, and extracellular polysaccharides (EPS). Exopolysaccharides generally consist of modified monosaccharides and, tentatively, some non-carbohydrate substituents such as acetate, pyruvate, succinate, and phosphate.
Many EPS, such as glucans and fructans, are produced extracellularly by the activity of glycosyl- or fructosyl-transferases, respectively, which use the disaccharide sucrose as the main substrate and are secreted by microorganisms into the culture medium. These enzymes cleave sucrose in a first step, and afterwards transfer either glucose or fructose to a growing sugar polymer forming glucans or fructans. The remaining free sugar monomers are subsequently metabolized.
For commercial purposes, EPS are currently isolated from fermentation broths after separation of biomass by downstream processes such as alcoholic precipitation (Donot, F., Fontana, A., Baccou, J. C., & Schorr-Galindo, S., Carbohydrate Polymers, 87:951-962, 2012). Current downstream processing to separate EPS from active compounds in fermentation broths may be costly and degrade the active compounds. The synthesis of viscous biopolymers hinders the fermentation process (i.e., stirring and oxygenation) as well as the recovery of highly purified molecules. There is a need for a cost-effective, efficient method of separating viscous biopolymers from active compounds in fermentation broth while preserving the activity of these compounds.